Jian Xing Ma

Rpe65 is necessary for production of 11- cis-vitamin A in the retinal visual cycle

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65–/– mice are disorganized compared with those of Rpe65+/+ and Rpe65+/– mice. Rod function, as measured by electroretinography, is abolished in Rpe65–/– mice, although cone function remains. Rpe65–/– mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65–/– mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Unbalanced expression of VEGF and PEDF in ischemia-induced retinal neovascularization.

Retinal levels of vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF), an angiogenic inhibitor, were measured and correlated with the ischemia‐induced retinal neovascularization in rats. The retinas with neovascularization showed a 5‐fold increase in VEGF while 2‐fold decrease in PEDF, compared to the age‐matched controls, resulting in an increased VEGF/PEDF ratio. The time course of the VEGF/PEDF ratio change correlated with the progression of retinal neovascularization. Changes in the VEGF and PEDF mRNAs preceded their protein level changes. These results suggest that an unbalance between angiogenic stimulators and inhibitors may contribute to retinal neovascularization.

Decreased pigment epithelium-derived factor and increased vascular endothelial growth factor levels in pterygia.

Purpose. Pterygia are histologically composed of proliferating fibrovascular tissue. This study compared expression levels of an angiogenic inhibitor, pigment epithelium-derived factor (PEDF), in pterygia with those in normal corneal and conjunctival tissues. Methods. The normal human conjunctival and corneal tissues were obtained from surgery or from donor eyes without ocular diseases. Pterygia were excised by therapeutic surgery under a microscope. Pigment epithelium-derived factor and vascular endothelial growth factor (VEGF) were measured by Western blot analysis. Their cellular localizations were determined by immunohistochemistry. Results. Intensive PEDF immunostaining was detected in all the normal corneal and conjunctival samples analyzed, predominantly in the epithelium and endothelium of the cornea and in the epithelium of the limbus and conjunctiva. Under the same immunostaining conditions, pterygial samples showed negative or faint PEDF staining. In contrast, the same pterygial samples all showed intensive VEGF staining, predominantly in the epithelium and in blood vessels. Western blot analysis confirmed that the average PEDF level in pterygia was drastically lower than those in normal corneal and conjunctival tissues, respectively. In contrast, the VEGF level in pterygia was significantly higher than in the normal tissues. Conclusion. Pterygia exhibit significantly lower PEDF but higher VEGF levels than those in normal corneas and conjunctivae. The decreased PEDF level in pterygia may play a role in the formation and progression of pterygia.

A Visual Pigment Expressed in Both Rod and Cone Photoreceptors

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

11-cis-Retinal Reduces Constitutive Opsin Phosphorylation and Improves Quantum Catch in Retinoid-deficient Mouse Rod Photoreceptors

Rpe65 −/− mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65 −/− mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65 −/− mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65 −/−mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.

Salamander UV cone pigment: Sequence, expression, and spectral properties

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

KALLISTATIN IN HUMAN OCULAR TISSUES : REDUCED LEVELS IN VITREOUS FLUIDS FROM PATIENTS WITH DIABETIC RETINOPATHY

PURPOSE Kallistatin is a serine proteinase inhibitor, which binds to tissue kallikrein and inhibits its proteolytic activity. This study is to determine the expression, cellular localization and the potential function of kallistatin in the eye. METHODS Tissue kallikrein-kallistatin complex formation was performed to detect the kallikrein-binding activity in ocular tissues. Immunoreactive kallistatin was detected and quantified by an enzyme-linked immunosorbent assay using polyclonal antibody specific to human kallistatin. In situ hybridization histochemistry was employed to localize the kallistatin mRNA in human eyes using an antisense riboprobe of kallistatin. RESULTS We have identified active kallistatin in the cornea, ciliary body, sclera, choroid, optic nerve, retina, vitreous and aqeous fluids. Kallistatin binds to tissue kallikrein and forms an SDS-stable complex. Immunoreactive kallistatin was identified in these tissues. Linear dose-dependent curves of the tissue extracts of the retina and choroid are parallel to that of purified human kallistatin, suggesting their immunological identity. The kallistatin mRNA was identified in the ciliary muscle, lens epithelial cells, all the layers of retina cells, optic nerve, choroid and vascular endothelial cells. These cells were not stained by the sense riboprobe under the same conditions, indicating the specificity of the hybridization. We also compared immunoreactive kallistatin levels in vitreous fluids from 18 patients with diabetic retinopathy and 17 non-diabetic subjects. The results show that diabetic subjects have significantly lower kallistatin levels (233.0 +/- 14.6 ng/mg protein) compared to non-diabetic subjects (334.1 +/- 26.9 ng/mg protein). CONCLUSIONS Kallistatin is produced endogenously in the eye and the decrease in the vitreous kallistatin levels may be involved in diabetic retinopathy.

Tissue Kallikrein-binding Protein Reduces Blood Pressure in Transgenic Mice

The kallikrein-kinin system participates in blood pressure regulation. One of the kallikrein-kinin system components, kallikrein-binding protein, binds to tissue kallikrein and inhibits its activity in vitro. To investigate potential roles of rat kallikrein-binding protein (RKBP) in vivo, we have developed transgenic mice that express an RKBP gene under the control of the mouse metallothionein metal-responsive promoter. Expression of the transgene, RKBP, was detected in the liver, kidney, lung, heart, pancreas, salivary glands, spleen, brain, testis, and adrenal gland at the mRNA and protein levels. Systolic blood pressures of homozygous transgenic mice were 88.5 ± 0.8 mm Hg (mean ± S.E., n = 19, P < 0.001) for one line and 88.8 ± 1.6 mm Hg (mean ± S.E., n = 19, P < 0.001) for another, as compared with 100.5 ± 0.8 mm Hg (mean ± S.E., n = 18) for control mice. Direct blood pressure measurements of these transgenic mice through an arterial cannula showed similar reductions of blood pressure. Intravenous injection of purified RKBP into mice via a catheter produced a dose-dependent reduction of the mean arterial blood pressure. Our findings suggest that RKBP may function as a vasodilator in vivo, independent of regulating the activity of tissue kallikrein.

Identification of RPE65 in transformed kidney cells1

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS‐1 and COS‐7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS‐7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full‐length encoding region was amplified from HEK293 and COS‐7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS‐7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.